Blood coagulation is a process consisting of a complex interaction of various blood components (or factors) that eventually gives raise to a fibrin clot. Generally, the blood components, which participate in what has been referred to as the coagulation “cascade”, are enzymatically inactive proteins (proenzymes or zymogens) that are converted to proteolytic enzymes by the action of an activator (which itself is an activated clotting factor). Coagulation factors that have undergone such a conversion are generally referred to as “active factors”, and are designated by the addition of the letter “a” to the name of the coagulation factor (e.g. Factor VIIa).
Initiation of the haemostatic process is mediated by the formation of a complex between tissue factor, exposed as a result of injury to the vessel wall, and Factor VIIa. This complex then converts Factors IX and X to their active forms. Factor Xa converts limited amounts of prothrombin to thrombin on the tissue factor-bearing cell. Thrombin activates platelets and Factors V and VIII into Factors Va and VIIIa, both cofactors in the further process leading to the full thrombin burst. This process includes generation of Factor Xa by Factor IXa (in complex with factor VIIIa) and occurs on the surface of activated platelets. Thrombin finally converts fibrinogen to fibrin resulting in formation of a fibrin clot.
Factor VII is a trace plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is catalytically inactive. Single-chain Factor VII may be converted to two-chain Factor VIIa by Factor Xa, Factor XIIa, Factor IXa, Factor VIIa or thrombin in vitro. Factor Xa is believed to be the major physiological activator of Factor VII. The conversion of zymogen Factor VII into the activated two-chain molecule occurs by cleavage of an internal Arg152-IIe153 peptide bond.
It is often desirable to stimulate or to selectively block the coagulation cascade in a subject. Factor VIIa has been used to control bleeding disorders that have several causes such as clotting factor deficiencies (e.g. haemophilia A and B or deficiency of coagulation Factors XI or VII) or clotting factor inhibitors. Factor VIIa has also been used to control excessive bleeding occurring in subjects with a normally functioning blood clotting cascade (no clotting factor deficiencies or inhibitors against any of the coagulation factors). Such bleeding may, for example, be caused by a defective platelet function, thrombocytopenia or von Wille-brand's disease. Bleeding is also a major problem in connection with surgery and other forms of tissue damage.
European Patent No. 200,421 (ZymoGenetics) relates to the nucleotide sequence encoding human Factor VII and the recombinant expression of Factor VII in mammalian cells.
Dickinson et al. (Proc. Natl. Acad. Sci. USA 93, 14379–14384, 1996) relates to Factor VII polypeptides wherein Lys 157, Val158, Glu296, Met298, Asp334, Ser336 or Lys337 have been individually replaced by Ala. Iwanaga et al. (Thromb. Haemost. (supplement august 1999), 466, abstract 1474) relates to Factor VIIa variants wherein residues 316–320 are deleted or residues 311–322 are replaced with the corresponding residues from trypsin.
Anticoagulants such as heparin, coumarin, derivatives of coumarin, indandione derivatives, or other agents may be used to selectively block the coagulation cascade in a patient, for example, during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), and a host of other medical disorders. For example, heparin treatment or extracorporeal treatment with citrate ion (U.S. Pat. No. 4,500,309) may be used in dialysis to prevent coagulation during the course of treatment. Heparin is also used in preventing deep vein thrombosis in patients undergoing surgery.
Treatment with heparin and other anticoagulants may, however, have undesirable side effects. Available anticoagulants generally act throughout the body, rather than acting specifically at a clot site. Heparin, for example, may cause heavy bleeding. Furthermore, with a half-life of approximately 80 minutes, heparin is rapidly cleared from the blood, necessitating frequent administration. Because heparin acts as a cofactor for antithrombin II (AT III), and AT III is rapidly depleted in DIC treatment, it is often difficult to maintain the proper heparin dosage, necessitating continuous monitoring of AT III and heparin levels. Heparin is also ineffective if AT III depletion is extreme. Further, prolonged use of heparin may also increase platelet aggregation and reduce platelet count, and has been implicated in the development of osteoporosis. Indandione derivatives may also have toxic side effects.
In addition to the anticoagulants briefly described above, several naturally occurring proteins have been found to have anticoagulant activity. For example, Reutelingsperger (U.S. Pat. No. 4,736,018) isolated anticoagulant proteins from bovine aorta and human umbilical vein arteries. Maki et al. (U.S. Pat. No. 4,732,891) disclose human placenta-derived anticoagulant proteins. In addition, AT III has been proposed as a therapeutic anticoagulant (Schipper et al., Lancet 1 (8069): 854–856 (1978); Jordan, U.S. Pat. No. 4,386,025; Bock et al., U.S. Pat. No. 4,517,294).
In about 30% or more of patients treated by angioplasty, endarterectomy or bypass grafts, thrombosis and/or smooth muscle cell proliferation in the intima causes re-occlusion of the vessel and consequent failure of the reconstructive surgery. This closure of the vessel subsequent to surgery is known as restenosis. Restenosis is thought to result from a complex interaction of biological processes including platelet deposition and thrombus formation, release of chemotactic and mitogenic factors, and the migration and proliferation of vascular smooth muscle cells into the intima of the dilated arterial segment.
The inhibition of platelet accumulation at sites of mechanical injury can limit the rate of restenosis in human subjects. While platelet accumulation occurs at sites of acute vascular injuries, the generation of thrombin at these sites may be responsible for the activation of the platelets and their subsequent accumulation.
International Application No. WO 92/15686 relates to inactivated Factor VIIa, polynucleic acid and mammalian cell lines for the production of inactivated Factor VIIa, and compositions comprising inactivated Factor VIIa for inhibiting blood coagulation.
International Application No. WO 94/27631 relates to methods for inhibiting vascular restenosis, tissue factor activity, and platelet deposition.
International Application No. WO 96/12800 relates to a method for treatment of acute closure of a coronary artery comprising to the individual a composition which comprises inactivated Factor VIIa in conjunction with tissue plasminogen activator or streptokinase.
Most proteins introduced into the circulation, are cleared quickly from the mammalian subject by the kidneys. This problem may be partially overcome by administering a larger amount of the protein or through repeated administration. However, higher doses of the protein can elicit antibodies which can bind and inactivate the protein and/or facilitate the clearance of the protein from the subject's body. Repeated administration of the therapeutic protein is essentially ineffective and can be dangerous as it can elicit an allergic response.
Various attempts to solve the problems associated with protein therapies include microencapsulation, liposome delivery systems, administration of fusion proteins, and chemical modification. The most promising of these to date is modification of the therapeutic protein by covalent attachment of polyalkylene oxide polymers, particularly polyethylene glycols (PEG). For example, U.S. Pat. No. 4,179,337 discloses the use of PEG or polypropylene glycol coupled to proteins to provide a physiologically active non-immunogenic water soluble polypeptide composition. Nucci et al. describe several proteins which have been modified by addition of PEG including adenosine deamidase, L-asparaginase, interferon alpha 2b (IFN-α2b), superoxide dismutase, streptokinase, tissue plasminogen activator (tPA), urokinase, uricase, hemoglobin, interleukins, interferons, TGF-beta, EGF, and other growth factors (Nucci et al., 1991, Adv. Drug Delivery Rev. 4:133–151). Attempts such as these have resulted in somewhat longer half-life of the proteins and reduction of protein immunogenicity.
Typically, PEGylation of proteins involves activating PEG with a functional group which will react with lysine residues on the surface of the protein. If the modification of the protein goes to completion, the activity of the protein is usually lost. Modification procedures which allow partial PEGylation of the protein usually result in only about 50% loss of activity and greatly increased serum half-life, so that the overall effective dose of the protein is lower.
Recent developments in protein PEGylation methods employ activated PEG reagents which react with thiol groups of the protein, resulting in covalent attachment of PEG to a cysteine, which residue was inserted in place of a naturally occurring lysine of the protein. Shaw et al. (U.S. Pat. No. 5,166,322) describe specific variants of IL-3 which have a cysteine introduced at specific sites within the naturally occurring amino acid sequence. Sulfhydryl reactive compounds (e.g. activated polyethylene glycol) are then attached to these cysteines by reaction with the IL-3 variant. Katre et al. (U.S. Pat. No. 5,206,344) describe specific IL-2 variants which contain a cysteine introduced at a specific site within the naturally occurring amino acid sequence. The IL-2 variant is subsequently reacted with an activated polyethylene glycol reagent to attach this moiety to a cysteine.
There is still a need in the art for improved Factor VII polypeptides having prolonged procoagulant or anticoagulant activity. In particular, there is a need for Factor VII polypeptides which has increased serum half-life without the undesirable side effects such as systemic activation of the coagulation system and bleeding, respectively, associated with conventional therapies, and which can be administered at relatively low doses so that repeated administrations of a larger amount of the protein are avoided.